PMID- 29321306 OWN - NLM STAT- MEDLINE DCOM- 20180411 LR - 20211204 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 92 IP - 6 DP - 2018 Mar 15 TI - Digitoxin Suppresses Human Cytomegalovirus Replication via Na(+), K(+)/ATPase alpha1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation. LID - 10.1128/JVI.01861-17 [doi] LID - e01861-17 AB - Host-directed therapeutics for human cytomegalovirus (HCMV) requires elucidation of cellular mechanisms that inhibit HCMV. We report a novel pathway used by cardiac glycosides to inhibit HCMV replication: induction of AMP-activated protein kinase (AMPK) activity and autophagy flux through the Na(+),K(+)/ATPase alpha1 subunit. Our data illustrate an intricate balance between the autophagy regulators AMPK, mammalian target of rapamycin (mTOR), and ULK1 during infection and treatment with the cardiac glycoside digitoxin. Both infection and digitoxin induced AMPK phosphorylation, but ULK1 was differentially phosphorylated at unique sites leading to opposing effects on autophagy. Suppression of autophagy during infection occurred via ULK1 phosphorylation at Ser757 by enhanced mTOR activity. Digitoxin continuously phosphorylated AMPK, leading to ULK1 phosphorylation at Ser317, and suppressed mTOR, resulting in increased autophagy flux and HCMV inhibition. In ATG5-deficient human fibroblasts, digitoxin did not inhibit HCMV, supporting autophagy induction as a mechanism for virus inhibition. Drug combination studies with digitoxin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) further confirmed the role of autophagy activation in HCMV inhibition. Individually, each compound phosphorylated AMPK, but their combination reduced autophagy rather than inducing it and was antagonistic against HCMV, resulting in virus replication. The initial ULK1 activation by digitoxin was counteracted by AICAR, which prevented the downstream interaction of Beclin1 and phosphatidylinositol 3-kinase class III (PI3K-CIII), further supporting digitoxin-mediated HCMV inhibition through autophagy. Finally, the alpha1 subunit was required for autophagy induction, since in alpha1-deficient cells neither AMPK nor autophagy was activated and HCMV was not inhibited by digitoxin. In summary, induction of a novel pathway (alpha1-AMPK-ULK1) induces autophagy as a host-directed strategy for HCMV inhibition.IMPORTANCE Infection with human cytomegalovirus (HCMV) creates therapeutic challenges in congenitally infected children and transplant recipients. Side effects and selection of resistant mutants with the limited drugs available prompted evaluation of host-directed therapeutics. We report a novel mechanism of HCMV inhibition by the cardiac glycoside digitoxin. At low concentrations that inhibit HCMV, digitoxin induced signaling through the alpha1 subunit of the Na(+),K(+)/ATPase pump and the cellular kinase AMPK, resulting in binding and phosphorylation of ULK1 (Ser317) and autophagy activation. HCMV suppressed autophagy through ULK1 phosphorylation (Ser757) by activating the mTOR kinase. The pump-autophagy pathway was required for HCMV inhibition, since in alpha1- or ATG5-deficient cells the virus was not inhibited. Furthermore, the AMPK activator AICAR antagonized digitoxin activity against HCMV, a phenomenon resulting from opposing effects downstream in the autophagy pathway, at the Beclin1 stage. In summary, autophagy may provide a strategy for harnessing HCMV replication. CI - Copyright (c) 2018 American Society for Microbiology. FAU - Mukhopadhyay, Rupkatha AU - Mukhopadhyay R AD - Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Venkatadri, Rajkumar AU - Venkatadri R AD - Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Katsnelson, Jenny AU - Katsnelson J AD - Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Arav-Boger, Ravit AU - Arav-Boger R AUID- ORCID: 0000-0002-5363-167X AD - Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA boger@jhmi.edu. LA - eng PT - Journal Article DEP - 20180226 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Intracellular Signaling Peptides and Proteins) RN - 0 (Ribonucleotides) RN - 360-97-4 (Aminoimidazole Carboxamide) RN - E90NZP2L9U (Digitoxin) RN - EC 2.7.1.1 (MTOR protein, human) RN - EC 2.7.11.1 (Autophagy-Related Protein-1 Homolog) RN - EC 2.7.11.1 (TOR Serine-Threonine Kinases) RN - EC 2.7.11.1 (ULK1 protein, human) RN - EC 2.7.11.31 (AMP-Activated Protein Kinases) RN - EC 3.6.1.- (ATP1A1 protein, human) RN - EC 7.2.2.13 (Sodium-Potassium-Exchanging ATPase) RN - F0X88YW0YK (AICA ribonucleotide) SB - IM MH - AMP-Activated Protein Kinases/genetics/*metabolism MH - Aminoimidazole Carboxamide/analogs & derivatives/pharmacology MH - Autophagy/*drug effects/genetics MH - Autophagy-Related Protein-1 Homolog/genetics/metabolism MH - Cells, Cultured MH - Cytomegalovirus/*physiology MH - Cytomegalovirus Infections/*drug therapy/genetics/metabolism/pathology MH - Digitoxin/*pharmacology MH - Fibroblasts/*metabolism/pathology/virology MH - Humans MH - Intracellular Signaling Peptides and Proteins/genetics/metabolism MH - Phosphatidylinositol 3-Kinases/genetics/metabolism MH - Ribonucleotides/pharmacology MH - Sodium-Potassium-Exchanging ATPase/genetics/*metabolism MH - TOR Serine-Threonine Kinases/genetics/metabolism MH - Virus Replication/*drug effects/genetics PMC - PMC5827378 OTO - NOTNLM OT - AMPK OT - ULK1 OT - autophagy OT - cardiac glycosides OT - cytomegalovirus EDAT- 2018/01/13 06:00 MHDA- 2018/04/12 06:00 PMCR- 2018/08/26 CRDT- 2018/01/12 06:00 PHST- 2017/10/25 00:00 [received] PHST- 2017/12/21 00:00 [accepted] PHST- 2018/01/13 06:00 [pubmed] PHST- 2018/04/12 06:00 [medline] PHST- 2018/01/12 06:00 [entrez] PHST- 2018/08/26 00:00 [pmc-release] AID - JVI.01861-17 [pii] AID - 01861-17 [pii] AID - 10.1128/JVI.01861-17 [doi] PST - epublish SO - J Virol. 2018 Feb 26;92(6):e01861-17. doi: 10.1128/JVI.01861-17. Print 2018 Mar 15.